The study of membrane proteins is important as the proteins represent 30% of cellular protein content and 70% of drug targets, and function as transporters, signal transduction mediators, and light harvesting centers, as well as electron transfer mediators in photosynthesis, among other key processes. Current techniques for membrane protein structure elucidation face obstacles due to difficulties in forming large crystals that are necessary for traditional X-ray crystallography. Smaller crystals form more easily, but they are destroyed by the high dose of radiation necessary to obtain adequate diffraction patterns and therefore cannot be used to obtain high quality structure information by traditional means. These issues are addressed by the development of femtosecond nanocrystallography in which X-ray exposure time is reduced to the femtosecond regime. Within these short time frames, nanocrystal X-ray damage is outrun so that diffraction patterns can be obtained before the crystal is destroyed.
In order to obtain high resolution diffraction patterns from crystals, a well-ordered crystal is necessary so that the diffracted signal is void of crystal lattice imperfections. Consequently, crystals in the sub-500 nm size regime are desired for improved shape transforms, crystal phasing uniformity, compatibility with beam diameters of the current state-of-the-art free electron lasers employed for nanocrystallography, and for compatibility with a jetting system used to introduce crystals to the beam. Variations in crystal size and shape lead to large amounts of single crystal diffraction data with several hundred thousand images needed for one data set. Obtaining a desired crystal size is difficult due to broad size distributions resulting from traditional crystallization, and moreover, first attempts to isolate nanocrystals such as gravitational settling procedures are time consuming and result in very low percent recoveries of desirably sized crystals.
Known nanoparticle sorting methods utilize centrifugation and filtration and result in a low abundance of protein nanocrystals, as sample loss and crystal fragmentation may occur. Other sorting methods employ conjugated or chemically functionalized nanoparticles for efficient separation yet are invasive to nanocrystallography and detrimental to downstream applications. Further, free-flow magnetophoresis methods may be suitable to separate nanoparticles continuously. However, methods based on free-flow magnetophoresis require that the nanoparticles have magnetic properties and thus cannot be applied to protein-based nanocrystals.